This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The first project focuses on the generation of discrete multi-protein assemblies through metal coordination chemistry. Despite extensive research, the ability to control protein-protein interactions (PPIs) remains a great challenge, owing to the fact that PPIs are guided by the superposition of many weak, non-covalent bonds spread over large surfaces. Our goal in this project is to utilize the strength, directionality and selectivity of metal-ligand interactions to control PPIs, thereby achieving specificity and affinity without requiring extensive binding surfaces. In preliminary experiments utilizing the four-helix bundle protein cytochrome cb562 as a building block, we have demonstrated that rationally designed metal-binding-motifs (MBMs) on protein surfaces can nucleate the formation of discrete multi-protein structures, whose oligomeric states and geometries are controlled entirely by metal coordination. Not only does this approach yield complex bioassemblies, but also gives rise to novel metallocenters built within protein-protein interfaces. We have so far collected crystallographic data sets on four superprotein assemblies, using a setup designed primarily for small molecule crystallography, which has yielded limited data resolution/quality. In this proposal, we aim to employ tunable synchrotron radiation to obtain high-resolution structures of up to 10 assemblies that we have crystallized, thoroughly establish the metal coordination geometries, and unambiguously confirm the presence of the metals contained within and determine their identities. The second project aims to map potential proton-transfer pathways in the molybdenum-iron protein (MoFeP) of nitrogenase, the enzyme responsible for biological nitrogen fixation. MoFeP catalyzes the 8 electron/8 proton reduction of dinitrogen into ammonia. While electron transfer in MoFeP is somewhat well understood, proton transfer pathways have not been established. Using an approach that has been successful with cytochrome c oxidase and